Development of protein therapy based on the application of cell membrane-permeable proteins has recently attracted considerable attention. As protein therapy, there is a method of constructing and administering a normal-type protein using an expression system, against a disease caused due to occurrence of abnormality, such as substitution or mutation, in amino acid sequences, resulting from frame shift or the like through the mutagenesis or recombination of a specific gene. Further, as shown in the recently reported construction of protein-induced pluripotent stem (piPS) cells, for induction of undifferentiated cells from already differentiated cells, there is a method of inducing undifferentiation by administering a cell membrane-permeable functional protein (4 types of transcription factors, Oct4, Klf4, Sox2, and cMyc) (for example, see Non-Patent Document 1).
Further, there is a need in regenerative medicine for the establishment of a method for differentiating a specific cell from undifferentiated cells, but it is also highly likely that a cell membrane-permeable protein will need to be applied thereto. Further, antibody therapy of inhibiting the function of a specific protein using an antibody is rapidly under development.
Conventionally, iPS cells have been established by introducing genes of 4 factors (Oct3/4, Sox2, Klf4, and c-Myc) into somatic cells, by means of a viral vector such as a retrovirus or lentivirus, but there has been concern about the risk of oncogenesis due to the insertion of a viral vector into a genome. In addition, the construction of a viral vector requires strictly controlled conditions which have been obstacles in the propagation of iPS cell techniques.
Accordingly, that a piPS cell can be produced by linking 11 arginines as a basic amino acid to 4 factors, modifying the linking product to have cell membrane permeability, and introducing the modified product into a mouse embryonic cell without using a virus has been published.
However, such a protein-based therapy has a disadvantage in that when a protein is administered to a cell or an individual, the protein is degraded by proteasome or phagosome, autophagy or proteolytic enzymes, or the like and, as a result, the administered protein may not particularly work, and may require very frequent administration (for example, see Non-Patent Document 1).
Currently, as a motif capable of inhibiting intracellular proteolysis of such a cell membrane-permeable protein, a poly-arginine tag is known. This is that 8R consisting of 8 arginines or a motif consisting mainly of arginine inhibits proteasomal proteolysis or the like (for example, see Non-Patent Documents 2 and 3).
Further, although it has been reported that an acidic amino acid motif is important for the stability of a protein (for example, see Non-Patent Documents 4 and 5), there is no report showing that an acidic amino acid motif is involved in the inhibition of proteolysis.
Even when an arginine-linked inducer is supplied to a piPS cell, since the inducer is susceptible to proteolysis, there are problems in that long-term administration of an inducer at a high volume into a cell should be made and the construction of a piPS cell takes a long period of several months.
Non-Patent Document 1: Zhou H. et al., Cell Stem Cell. 2009; 4(5): 381-384
Non-Patent Document 2: Kloss A. et al., Eur J Pharm Biopharm. 2009; 72(1): 219-225.
Non-Patent Document 3: Anbanandam A. et al., J Mol. Biol. 2008; 384(1): 219-227.
Non-Patent Document 4: Yi P. et al., Mol. Cell. 2008; 29(4): 465-476.
Non-Patent Document 5: Yen H C. et al., Science. 2008; 322(5903): 918-923.